Transmission efficiency of Xylella fastidiosa by sharpshooters (Hemiptera: Cicadellidae) in coffee and citrus

Xylella fastidiosa (Wells, Raju, Hung, Weisburg, Mandelco-Paul, and Brenner) is a bacterial pathogen transmitted by several sharpshooters in two tribes of Cicadellinae (Proconiini and Cicadellini). Here, we compared the transmission efficiency of X. fastidiosa in coffee (Coffea arabica L.) and citrus [Citrus sinensis (L.) Osbeck] by Cicadellini [Bucephalogonia xanthophis (Berg) and Dilobopterus costalimai Young] and Proconiini [Homalodisca ignorata Melichar and Oncometopia facialis (Signoret)] sharpshooters that occur in both crops. At different seasons, healthy adults of each species were submitted to a 48-h acquisition access period on citrus or coffee source plants infected with X. fastidiosa isolates that cause Citrus variegated chlorosis (CVC) and Coffee leaf scorch (CLS), respectively, and then confined on healthy seedlings of the corresponding host plant for a 48-h inoculation access period. No significant effect of inoculation season was observed when comparing infection rates of citrus or coffee plants inoculated by vectors at different times of the year. In citrus, the transmission rate by single insects was significantly higher for H. ignorata (30%) in relation to B. xanthophis (5%) and O. facialis (1.1%), but there was no difference among vector species in coffee, whose transmission rates ranged from 1.2 to 7.2%. Comparing host plants, H. ignorata was more effective in transmitting X. fastidiosa to citrus (30%) in relation to coffee (2.2%), whereas the other vectors transmitted the bacterium to both hosts with similar efficiencies. Despite these variations, vector efficiency in coffee and citrus is lower than that reported in other hosts.     

Physiological changes associated with senescence and abscission in mature citrus fruit induced by 5-chloro-3-methyl-4-nitro-1H-pyrazole and ethephon application

This research compares effects of the compound 5-chloro-3-methyl-4-nitro-1 H -pyrazole (CMNP), a plant growth regulator that selectively promotes abscission in mature citrus fruit ( Citrus sinensis ), and the ethylene-releasing agent ethephon (2-chloroethylphosphonic acid). Application of CMNP and ethephon to mature citrus fruit reduced fruit detachment force and changed peel color from green to orange. More total chlorophyll was extracted from flavedo in early season (November) than late season (January), and both compounds caused a similar reduction in chlorophyll. In contrast, total carotenoid content was similar in November and January. Both abscission compounds increased total carotenoids, but induction was greater in January, and CMNP was more effective in both months. Phospholipase A₂ (PLA₂) activity increased after CMNP but not ethephon application. Electrolyte leakage increased 2 h after CMNP treatment, and total protein content was reduced by 50% after 72 h. Ethephon caused only minor changes in electrolyte leakage and total protein content. Inhibition of PLA₂ activity with aristolochic acid did not reduce leakage but inhibited total protein loss and reduced visual peel damage associated with CMNP. Ultrastructural observations indicated decreased number, and length of starch grains 3 h after CMNP treatment. A transient increase in soluble sugars was measured 3 h after CMNP application. Ethephon had little effect on soluble sugar content and changes in starch grains. Collectively, the results indicate that CMNP and ethephon induced color change in peel and advanced mature fruit abscission. However, CMNP but not ethephon promoted other physiological changes associated with senescence.     

Helicoverpa armigera granulovirus interference with progression of H. zea nucleopolyhedrovirus disease in H. zea larvae

Capsular proteins from Helicoverpa armigera granulovirus (HaGV) have previously been shown to enhance H. armigera nucleopolyhedrovirus (HaSNPV) infection in H. armigera larvae. Yet, HaGV and HaS-NPV, as viable viruses, interfered with one another. In our study, we have examined the effects of co-infection of the slow-killing virus HaGV with the fast-killing virus Helicoverpa zea NPV (HzSNPV) on H. zea larvae. The mortality parameter measured was survival time. Virus stocks had 50% lethal concentrations of 3.2x10(-9) g HaGV-infected cadavers (GVC) (HaGV) and 32 occlusion bodies (HzSNPV) per cup. Average survival times were 16.8 and 5.5 days for larvae treated with HaGV and HzSNPV, respectively; death of HzSNPV-treated larvae was as early as 72 h posttreatment. In co-infection experiments in which larvae were treated concurrently with both viruses, the viruses competed in typical fashion for host resources. However, interference with disease progression in HzSNPV-fed larvae occurred even when HaGV was fed to larvae up to 36 h after the NPV, a time at which NPV infection should have been well established in host larvae. At death, co-infected larvae were observed microscopically to be filled with HaGV granules rather than HzSNPV polyhedra. The time study results imply that HaGV might be outcompeting HzSNPV by inhibiting its replication. We also observed that H. zea larvae treated with high dosages of HaGV (> or =3x10(-5) g GVC) were initially stunted but had survival times similar to those of larvae treated with lower dosages.     

Host status of grapefruit and Valencia oranges for Anastrepha serpentina and Anastrepha ludens (Diptera: Tephritidae)

Anastrepha serpentina (Wiedemann) (Diptera: Tephritidae) is sporadically captured in the Rio Grande Valley of Texas. Although its preferred hosts are in the Sapotaceae family, several varieties of Citrus, including grapefruit and oranges are listed as alternate hosts. Although Mexican fruit fly, Anastrepha ludens (Loew), is known to be a major pest of Citrus, doubt exists as to the status of Citrus as a breeding host for A. serpentina. To evaluate the host status of commercial Citrus for A. serpentina we compared oviposition and development with that of A. ludens under laboratory conditions with 'Rio Red' grapefruit (Citrus paradisi MacFayden) and 'Valencia' oranges [Citrus sinensis (L.) Osbeck] in different stages of maturity. Both fly species oviposited in early season fruit in which the eggs and larvae died in the fruit albedo. Survival of either species to the adult stage occurred in later season grapefruit. In oranges, no A. serpentina larvae survived compared with 150 A. ludens surviving to adults. Survival on both Citrus species was much lower for A. serpentina, only approximately 5% of eggs eclosed into larvae in grapefruit compared with approximatley 50% for A. ludens. In oranges approximately 16% of A. serpentina eggs eclosed compared with approximately 76% for A. ludens. In grapefruit, only one fourth as many A. serpentina larvae survived to the adult stage compared with A. ludens. Additional experiments were performed in a greenhouse on small, caged trees of la coma (Sideroxylon celastrinum H.B.K.), a Texas species of Sapotaceae. The A. serpentina females readily oviposited into these berries and normal adults emerged. The present low incidence of the adults, coupled with the high mortality during development of the larvae, suggests that Texas citrus is unlikely to support a breeding population of A. serpentina.     

Cross-pollination of nontransgenic corn ears with transgenic Bt corn: efficacy against lepidopteran pests and implications for resistance management

The efficacy of nontransgenic sweet corn, Zea mays L., hybrids cross-pollinated by Bacillus thuringiensis (Bt) sweet corn hybrids expressing Cry1Ab toxin was evaluated in both field and laboratory studies in Minnesota in 2000. Non-Bt and Bt hybrids (maternal plants) were cross-pollinated with pollen from both non-Bt and Bt hybrids (paternal plants) to create four crosses. Subsequent crosses were evaluated for efficacy in the field against European corn borer, Ostrinia nubilalis (Hübner), and corn earworm, Helicoverpa zea (Boddie), and in laboratory bioassays against O. nubilalis. Field studies indicated that crosses with maternal Bt plants led to low levels of survival for both O. nubilalis and H. zea compared with the non-Bt x non-Bt cross. However, the cross between non-Bt ears and Bt pollen led to survival rates of 43 and 63% for O. nubilalis and H. zea larvae, respectively. This intermediate level of survival also was reflected in the number of kernels damaged. Laboratory bioassays for O. nubilalis, further confirmed field results with larval survival on kernels from the cross between non-Bt ears and Bt pollen reaching 60% compared with non-Bt crossed with non-Bt. These results suggest that non-Bt refuge plants, when planted in proximity to Bt plants, and cross-pollinated, can result in sublethal exposure of O. nubilalis and H. zea larvae to Bt and may undermine the high-dose/refuge resistance management strategy for corn hybrids expressing Cry1Ab.     

哈姆林甜橙与粗柠檬体细胞杂种的育性

对异源四倍体柑桔体细胞杂种“哈姆林甜橙+粗柠檬”及其亲本的花粉活力、花器官发育、花器官形态发生与花粉母细胞减数分裂四分体阶段进行了观测和统计.结果发现“哈姆林甜橙+粗柠檬”的花粉染色活力、萌发率、每花药中花粉粒数均居于其双亲之间,花器官发育及其形态发生具有双亲的特点.但小花粉及花粉母细胞减数分裂过程中形成的不正常四分体比率远远高于其双亲.以体细胞杂种“哈姆林甜橙+粗柠檬”为花粉亲本,与二倍体单胚类型宜昌橙与华农本地早的有性后代杂交,获得了110棵有性后代植株,其中三倍体82棵,二倍体和其它倍性的植株28棵.     

Central role of hemocytes in Autographa californica M nucleopolyhedrovirus pathogenesis in Heliothis virescens and Helicoverpa zea

Autographa californica M nucleopolyhedrovirus (AcMNPV) can infect and kill a wide range of larval lepidopteran hosts, but the dosage required to achieve mortal infection varies greatly. Using a reporter gene construct, we identified key differences between AcMNPV pathogenesis in Heliothis virescens and Helicoverpa zea, a fully permissive and a semipermissive host, respectively. Even though there was more than a 1,000-fold difference in the susceptibilities of these two species to mortal infection, there was no significant difference in their susceptibilities to primary infections in the midgut or secondary infections in the tracheal epidermis. Foci of infection within the tracheal epidermis of H. zea, however, were melanized and encapsulated by 48 h after oral inoculation, a host response not observed in H. virescens. Further, H. zea hemocytes, unlike those of H. virescens, were highly resistant to AcMNPV infection; reporter gene expression was observed only rarely even though virus was taken up readily, and nucleocapsids were transported to the nucleus. Collectively, these results demonstrated that hemocytes-by removing virus from the hemolymph instead of amplifying it and by participating in the encapsulation of infection foci-together with the host's melanization response, formed the basis of H. zea's resistance to fatal infection by AcMNPV.     

Citrus Leprosis Virus Vectored by Brevipalpus phoenicis (Acari: Tenuipalpidae) on Citrus in Brazil

Citrus leprosis is caused by Citrus leprosis virus (CiLV) that is transmitted by mites in the genus Brevipalpus (Acari: Tenuipalpidae). This disease directly reduces production and the life span of the citrus plant. The main symptoms of the disease include lesions on fruits, leaves, and twigs or small branches, causing premature fruit drop, defoliation, and death of the twigs or branches leading to serious tree decline. Leprosis is a highly destructive disease of citrus, wherever it occurs. The Brazilian citrus industry spends over 100 million US dollars annually on acaricides to control the vector, Brevipalpus phoenicis (Geijskes). This review contains information about the history of the etiology of citrus leprosis, its geographical distribution, host range, the role of the mite vectors, viral morphology and relationships with the infected cell, and transmissibility of the virus by the mite. In addition, data on the mite-virus-plant relationship, disease damage, and strategies for controlling disease spread are presented.     

Field evaluation of a Helicoverpa zea (Lepidoptera: Noctuidae) damage simulation model: effects of irrigation, H. zea density, and time of damage on cotton yield

Helicoverpa zea (Boddie) is an important pest of cotton, Gossypium hirsutum L., for which many economic injury and population models have been developed to predict the impact of injury by this species on cotton yield. A number of these models were developed using results from simulated damage experiments, despite the fact that no studies have demonstrated that simulated damage is comparable to real H. zea damage. Our main objective in this study was to compare the effect on yield of H. zea larvae feeding on cotton fruiting structures at different irrigation levels, larval densities, and cotton physiological ages with damage produced artificially by removing fruiting structures by hand using simulated estimates of H. zea injury. To accomplish this, we used two irrigation levels, each divided into real and simulated damage plots. In real damage plots, H. zea larvae were placed on plants and allowed to feed; whereas in simulated damage plots, fruiting structures were removed by hand using a simulation model of H. zea damage to determine numbers and amounts of fruiting structures to remove. Each of these plots was further divided into one undamaged control plot and nine treatment plots. Each treatment plot was randomly assigned one of three damage times (early, middle, or late season) and one of three H. zea densities. In 1998, we found that only artificial H. zea damage (performed by hand removal of fruiting structures) at the highest density and during the late season decreased yield; whereas real damage caused by H. zea larvae placed on plants, and artificial damage occurring at earlier time periods and lower H. zea densities did not affect yield. In 1999, both real and artificial damage decreased yield at the higher H. zea densities compared with the lowest density, but, as in 1998, this was only true when damage occurred late in the season. The most important finding of this study was that high H. zea densities had no effect on cotton yield unless they occurred late in the season. In particular, this was true for artificial H. zea damage. The second most important finding of this study was that, with the exception of late in the season, our model for simulating H. zea damage to cotton through removal of fruiting structures resulted in similar yields as real H. zea larvae damage to cotton.     

Field deposition of Bt transgenic corn pollen: lethal effects on the monarch butterfly

We present the first evidence that transgenic Bacillus thuringiensis (Bt) corn pollen naturally deposited on Asclepias syriaca; common milkweed, in a corn field causes significant mortality of Danaus plexippus L. (Lepidoptera: Danaidae) larvae. Larvae feeding for 48 h on A. syriaca plants naturally dusted with pollen from Bt corn plants suffered significantly higher rates of mortality at 48 h (20Dž%) compared to larvae feeding on leaves with no pollen (3Dž%), or feeding on leaves with non-Bt pollen (0%). Mortality at 120 h of D. plexippus larvae exposed to 135 pollen grains/cm² of transgenic pollen for 48 h ranged from 37 to 70%. We found no sub-lethal effects on D. plexippus adults reared from larvae that survived a 48-h exposure to three concentrations of Bt pollen. Based on our quantification of the wind dispersal of this pollen beyond the edges of agricultural fields, we predict that the effects of transgenic pollen on D. plexippus may be observed at least 10 m from transgenic field borders. However, the highest larval mortality will likely occur on A. syriaca plants in corn fields or within 3 m of the edge of a transgenic corn field. We conclude that the ecological effects of transgenic insecticidal crops need to be evaluated more fully before they are planted over extensive areas.     

Pollen layering and male–male competition: Quantum dots demonstrate that pollen grains compete for space on pollinators

Premise

Almost nothing is known about what happens to pollen grains once they attach to pollinators, although some have postulated that pollen from different donors may form complex, two- or three-dimensional landscapes (e.g., layers or mosaics) that can facilitate male–male competition. For example, pollen that is already on pollinators may preclude the deposition of subsequent pollen grains.

Methods

Using quantum dots to mark the pollen of individual flowers, we explored the possibilities of layering and preclusion in a fly-pollinated iris, Moraea lurida.

Results and Conclusions

The proportion of labeled pollen from the last flower visited diminished in sequential pollen samples taken from the top to the bottom of the pollen load, representing the first empirical evidence for pollen layering. However, the consequences in terms of pollen preclusion were equivocal: Although the pre-existing pollen load size was not a good predictor of new pollen receipt, labeled pollen loads from the last flower visited were significantly smaller than pollen loads from the previous flower visited. Thus, pollen from the previous flower may preclude pollen placement from a subsequently visited flower, and pollen from different flowers may compete for space on pollinators.

     

A New Secondary Pollen Presentation Mechanism from a Wild Ginger (Zingiber densissimum) and Its Functional Roles in Pollination Process

Background and Aims

Secondary pollen presentation (SPP), a floral mechanism of reproductive adaptation, has been described for more than 200 years, with nine types SPP recorded. However, few studies have been done experimentally to link the floral mechanism of SPP to its functional roles in pollination process. This study aims to describe a new SPP mechanism from a wild ginger (Zingiber densissimum, Zingiberaceae) and explore how the pollen arrangement of SPP affects pollen removal during the interaction with different pollinators.

Methodology/Principal Findings

Field observations and experiments revealed that flowers lasted for less than one day. The breeding system was partially self-incompatible. Two bee species, Macropis hedini (which carried pollen dorsally) and Amegilla zonata (which carried pollen ventrally) were the primary pollinators. About a third of pollen grains were relocated from the anther to the labellum staminode of flowers through the adherence of aggregated pollen chains, while other grains were presented on the anther. In a single visit, each bee species removed pollen grains from both the labellum staminode and the anther. Macropis hedini was more effective than Amegilla zonata.

Conclusions/Significance

Our study describes a new SPP mechanism in angiosperms. The new SPP mode enables pollen grains presented on the anther and the labellum staminode simultaneously via the adherence of aggregated pollen chains, thus promoting pollen to be taken away by different pollinators. This SPP mechanism plays a key role during pollen removal and may have evolved under the pressure to improve male fitness.     

Generation of reactive oxygen species during pollen grain germination

The formation of reactive oxygen species in pollen at the early germination stage, which precedes the formation of the pollen tube, was studied. During this period, pollen grain is being hydrated, abruptly increasing its volume, and it passes from the resting state to active metabolism. Fluorescent methods have made it possible to reveal reactive oxygen species in the cytoplasm and inner layer of the pollen wall, intine. The cytoplasmic reactive oxygen species were mostly found in mitochondria, while extracellular ones were localized in aperture zones of intine, as well as in the solution surrounding pollen grains in vitro. The content of extracellular reactive oxygen species decreased after superoxide dismutase (100 units per ml) and diphenylene iodonium (100 μM), which indicates NADPH oxidase as one of possible producent of them. In conditions of suppression of extracellular reactive oxygen species production (100 μM diphenilene iodonium) or their promoted removal (after addition of 10 to 100 μM ascorbic acid), the number of germinating pollen grains increased. This effect disappeared after further increase in the concentration of the listed reagents. The result is evidence of the significance of processes of generation/removal of extracellular reactive oxygen species for pollen germination.     

沙田柚黄龙病病原体的电镜观察与PCR检测

柑桔黄龙病可以侵染沙田柚而产生典型的‘斑驳’症状。我们通过电镜观察表明,带病沙田柚的病原体在形态,大小和构造上均与柑桔黄龙病病原体（类细菌）相似：PCR扩增也证明其病原体DNA与柑桔黄龙病病原体的PCR产物具有很高的同源性。     

Influence of the host plant on occluded virus production and lethal infectivity of a baculovirus

Intra- and inter-specific effects of cotton, soybean, and clover on the time until death of Helicoverpa zea (Boddie) and Heliothis virescens (F.) larvae lethally infected with H. zea nucleopolyhedrovirus (HzSNPV) were evaluated in the laboratory. In the first test, on second instar only, the time until death of lethally infected larvae of both species differed with the plant tissues (vegetative or reproductive) and plant species. The total viral activity produced per larva in LC(50) units (occluded viral bodies (OBs) per larva/LC(50) in OBs/mm(2) of diet surface) was greater from H. virescens larvae fed vegetative than reproductive tissues of all host plants, but from H. zea virus production was greater only when fed vegetative tissue of soybean. In a second test that compared second and fourth instar H. virescens on cotton, total viral activity from larvae treated in both instars was greater when fed vegetative than reproductive tissues. Results of these tests suggest that the ability of host plants to influence baculovirus disease is more complex than previously believed. When examining the epizootic potential of a baculovirus, more attention must be given to the effects of the host plant on the insect-virus interactions.     

In vitro pollen germination in Hypericum perforatum L. and Hypericum rumeliacum Boiss

In vitro pollen germination and pollen tube growth investigations are valuable tools used in identification of the effects of environmental factors and genotypic differences on pollen viability, pollen germination and tube elongation. In this study pollen viability, in vitro pollen germination capacity, abnormality ratios and tube length in germinated pollens of Hypericum perforatum L. and H. rumeliacum Boiss. were investigated. Both of these species has spheroid-shaped and tricolporate pollen grains. The diameters of Hypericum perforatum and H. rumeliacum pollens were found as 24 +/- 3 microm and 19 +/- 2 microm, respectively. Pollen viability of H. perforatum and H. rumeliacum was found as 83% and 72%, respectively. The germination percentages were found as 12.85% for H. perforatum and 64.42% for H. rumeliacurm. Tube lengths in germinated pollens of both taxa were measured approximately as 95.25 +/- 38 microm in H. perforatum and 165.92 +/- 53 microm in H. rumeliacium 4 h after inoculation. In germinated pollen grains of H. perlbratum and H. rumeliacumn abnormality percentages were determined as 13.23% and 43.97%, respectively. In germinated pollens of these two species, highly significant (P < 0.00001) differences in in vitro germination percents and abnormality percents were observed. Abnormalities such as swollen tube tip, branched tube, spiralled tube and excessive tube formation were observed in pollen tubes. The results of this study showed that there were obvious differences in pollen germinability between these two species growing under the same environmental conditions.     

Flow cytometric analysis of pollen grains collected from individual bees provides information about pollen load composition and foraging behaviour

Background and Aims

Understanding the species composition of pollen on pollinators has applications in agriculture, conservation and evolutionary biology. Current identification methods, including morphological analysis, cannot always discriminate taxa at the species level. Recent advances in flow cytometry techniques for pollen grains allow rapid testing of large numbers of pollen grains for DNA content, potentially providing improved species resolution.

Methods

A test was made as to whether pollen loads from single bees (honey-bees and bumble-bees) could be classified into types based on DNA content, and whether good estimates of proportions of different types could be made. An examination was also made of how readily DNA content can be used to identify specific pollen species.

Key Results

The method allowed DNA contents to be quickly found for between 250 and 9391 pollen grains (750–28 173 nuclei) from individual honey-bees and between 81 and 11 512 pollen grains (243–34 537 nuclei) for bumble-bees. It was possible to identify a minimum number of pollen species on each bee and to assign proportions of each pollen type (based on DNA content) present.

Conclusions

The information provided by this technique is promising but is affected by the complexity of the pollination environment (i.e. number of flowering species present and extent of overlap in DNA content). Nevertheless, it provides a new tool for examining pollinator behaviour and between-species or cytotype pollen transfer, particularly when used in combination with other morphological, chemical or genetic techniques.     

The digestion of dandelion pollen by adult worker honeybees

ABSTRACT. The digestion of dandelion ( Taraxacum officinale ) pollen by adult worker honeybees ( Apis mellifera Linn.; Hymenoptera: Apidae) was initiated at the germination pores. 30 min after feeding, pollen had reached the anterior midgut and the germination pores had become swollen. This permitted further removal of protoplasm during the next 2 h of digestion as the pollen passed into the middle portion of the midgut. 3 h after feeding, pollen grains had reached the posterior midgut where some had ruptured to release both 'naked' protoplasm and masses of protoplasm but many remained intact or were only partially digested; undigested pollen grains passed unchanged to the rectum. The lipid-rich pollenkitt layer was removed from the exine during digestion. Our studies indicate that dandelion pollen was not utilized completely by honeybees.